1. Technical Field of the Invention
The present invention relates, generally, to a process for the preparation and use of chemotherapeutic agents.
More particularly, the present invention relates to a process for the preparation of high purity nucleoprotamine-DNA complex substances and a process for their use as an anti-tumor or anti-viral agent, including their use as an anti-AIDS agent. Additionally, research data exists to further suggest that certain aging characteristics, in test animals, might be slowed, or even reversed, with the foregoing chemical agents.
The nucleoprotamine-DNA complex substances are also useful in a variety of other medical conditions, some of which are serious, in humans and other mammals. Elevated serum cholesterol levels have also responded favorably to treatment.
2. Description of the Prior Art
Nucleohistones have been generally known to the art to be closely associated with DNA, and research evidence exists to suggest that such substances protect DNA by wrapping about the double helix of the DNA in adult cells. Hnilica, Lubomir S., The Structure and Biological Functions of Histones, p. 37 (CRC Press 1972). By contrast, nucleoprotamines are physically associated with the DNA of embryonic tissue and are not found in adult cells.
Nucleohistones and nucleoprotamines, however, do possess several common characteristics: Both are generally low molecular weight polypeptides (&lt;30,000 Dalton), rich in the amino acid arginine, slowly soluble in water, and resistant to heat coagulation. Both histones and protamines have overall positive charges and are in the basic range of pH. Additionally, both are bound to the negative charge of DNA, with known affinity constants, and both are shielded by the negative charge of DNA. Histones are known to be soluble in very dilute mineral acids, but insoluble in mild aqueous NH.sub.4 OH. By contrast, protamines are soluble in both very dilute mineral acids and mild aqueous NH.sub.4 OH.
In the past several years, various research investigators have suggested that histones, and possibly protamines and protamine-like polypeptides, exert a control function over DNA through a direct physical contact, or a lack thereof, at a myriad of sites along the DNA in the genome of all living cells. This direct physical contact at the molecular level constitutes charge cloud interactions between the proteins and the deoxyribose background. The isolation of only a few histone and protamine subunits, and the monotony of their amino acid sequences in different tissues from the same animal, and even from different animals, has suggested that histones and protamines act as merely a protective wrapper for the cellular DNA and lack the expected variability in their amino acid sequence to control transcription of messenger RNA (mRNA).
Furthermore, the structural theories about DNA have suggested that DNA helices have a major and minor groove along the alpha-helix. DNA bases appear to be in the bottom of the major groove, and the deoxyribose backbone in the bottom of the minor groove. Speculation that sequence specific proteins may attach at either the minor or major groove sites, along with histone and protamines or protaminelike proteins, to control transcription or DNA, has been widely accepted. See, Li, Hsueh Jei, Chromatin and Chromosome Subunits, Academic Press, New York (1977).
Simple systems for the control of DNA expression are also well known to the prior art, such as the Lactose Operon (LAC Operon) system of prokaryotic cells. An analysis of this operon model illustrates the concept of repressors and inducers as being fundamental control systems for mRNA transcription. See, e.g., Kim, R., and S. H. Kim, "Direct measurement of DNA unwinding angle in specific interaction between lac operator and repressor." Cold Spring Harbor Symp. Quant. Biol., 47: 481-484 (1983); Wang, J., M. D. Barkley, and S. Bourgeois, "Measurements of unwinding of lac operator by repressor." Nature, 251: 247-249 (1974).
In the LAC Operon, a promoter site on the DNA is followed by an operator site and structural gene sequences for three enzymes required for the hydrolysis and control of the galactose to glucose metabolic pathway. Galactose, along with catabolite activator protein (CAP), cyclic AMP (c-AMP), and RNA polymerase are capable of acting as an inducer, displacing the LAC repressor protein from the operator site, presumably accessable through the major groove, binding with the promoter site, and allowing transcription of the structural genes to proceed. If glucose is present, it acts to block the formation of the active inducer complex and the cell's own heterogeneous repressor remains attached to the operator site, with no transcription of the LAC genes possible.
Thus, by negative feedback inhibition, glucose controls the transcription of the LAC operon. Repressors, under this theory, have a negative influence on transcription, and this is an important aspect of control which the invention focuses upon.
By way of background, repressors may have developed, through evolution, as mutations, or acquired oncogenes, in very early unicellular promordial organisms. A mutant, with an incomplete repressor, may have had a competitive, if not at least a metabolic advantage, if it could halt the production of a protein when the protein was sufficiently abundant in the cytoplasm, and then re-start production of the protein as the need was developed in the cell. It is postulated that a mutant with an incomplete repressor would consume less energy than those of a normal phenotype and would be favored for survival under the theory of natural selection. Complete repressor mutants must have obviously died, if the synthesis of the particular protein so repressed was critical for survival. However, the incomplete repressor mutant could have survived if the repressor was only "loosely" attached to the operator site, and a cellular protein, or lack of such protein, influenced the repressor to "fall off" the particular operator site.
Now consider obligate parasites, such as a virus. For such parasites to have the ability to shut-down a host cell's transcription of proteins for the host cell, so that the "pirated" cellular machinery could be utilized to transcribe viral proteins, would yield such "life" forms a tremendous evolutionary advantage. Again, by evolution and mutation, viruses may have developed their own cell-directed repressors, encoded into viral DNA or RNA, transcribed when the virus DNA infected the host DNA, or translated from viral RNA, and subsequently pre-packaged with the viral genetic material during lysogeny phase in prokaryotic cells. Evidence that transcription of prokaryotic cellular proteins frequently ceases within minutes after viral infection, strongly supports this theory. Stryer, Lubert, Biochemistry, p. 712 (W. H. Freeman and Company, San Francisco, 1975.)
The infection of eukaryotic cells, by contrast, rarely leads to a total shutdown of host transcription, but rather, results in subtle repressor mediated subversion of both cytoplasmic and nuclear host process; possibly the next stage in the evolutionary process, avoiding a less energy efficient total shutdown.
Consider the specificity of the foregoing types of repressors, one of homogenetic cellular origin, and one, what is recognized by the cell to be, of allogenetic viral origin. The cell's repressor (C-rep) has evolved a very specific operator region to match its complementary operator site (e.g., only 27 base-pairs long, with some symmetry, in E. coli.), with matched base sequence by base pair to base pair in the operator region; a form of evolved primary structure, with a high rate constant of association (e.g., 7.times.10.sup.9 m.sup.-1 in E. coli.); and, other primary, secondary, tertiary and quarternary protein structural evolutions in the remainder of the specialized globular protein (approximately 30,000 Daltons) to interpret the various cytoplasmic signals that dictate to "release" or "remain attached." Stryer, Lubert, Biochemistry, p. 684 (W. H. Freeman and Company, San Francisco, 1975.)
Concerning the viral repressor (V-rep), originating from a viral DNA (or, in some cases, RNA) strand of small proportions (e.g., 10.sup.6 -10.sup.7 Daltons) (Stryer, Lubert, Biochemistry, p. 709 (W. H. Freeman and Company, San Francisco, 1975.)), it would be of great advantage to the viral repressor if it were to successfully complement the base pairing of the operator region in a number of host cells. This would be expected if the base pairing in the operon anticodon region of the V-rep was less specific than that of the C-rep. In short, viruses, and possibly other living organisms, have probably evolved poor fitting, but nonetheless effective repressors, when at an evolutionary advantage to do so. In fact, as discussed above, perfectly fitting repressors could conceivably act as complete repressors, thereby possibly having a lethal effect on the cell.
Consider, now, the situation presented when a host cell is under attack or otherwise infected by an assortment of viral agents and other life forms; poorly fitting allogenetic repressors, repressors evolved without the globular protein structure necessary for their timely removal at specific intracellular prompt conditions. Under such conditions, it is clear that the control of protein synthesis within the cell may be severely affected.
Now, reconsider the postulated evolutionary trends of repressors, but now allow for inducers, globlar proteins (or combinations of proteins) that greatly enhance m-RNA transcription rates, to also be imitated. Not only are host cells producing less of some proteins due to repression, but the host may actually begin to produce greater amounts of other proteins due to allogenetic inducers. False allogenetic repression and induction may completely disrupt a cell's metabolic process, and at the simplest level, the disruption of a cell's normal metabolic processes are the classic causes of cancer.
The general histological changes of tissue associated with the regression of a cell toward a cancer are known. Such cells are less differentiated, tend to function and appear as embryonic tissues and have been described as chaotic in their metabolic pathways and metastatic without regard to their proper location.
Thus, control of protein synthesis means proper health for a cell. Conversely, the lack of control or proper regulation of protein synthesis results in aberrant metabolism, dysfunction and sometimes even death of the cell.
The theory behind nucleoprotamine therapy states that the treatment of mammals with specifically timed collections of extracted nucleoprotamine and protamine-like proteins removes false repressors and false inducers, due to the lack of complete operon affinity in these heterogenetic proteins.
Additionally, consider an important adult tissue operon (a length of genetic coding sequence required to make a protein necessary for the health of the cell) that has been repressed by a repressor protein of allogentic origin, such a viral protein from a recent viral infection. Adaptation of Wilkin's 1956 model depicts an allogenetic repressor occupying the major groove of the DNA helix over an operator region. Wilkins, M. H. F., Physical Studies of the Molecular Structure of Deoxyribose Nucleic Acid and Nucleoprotein, Cold Spring Harbor Symposium Quantitative Biology, 21, 75-90 (1956). The allogenetic repressor is, in all likelihood, poorly physically bound to the operator region of the operon thereby physically preventing the attachment of the RNA polymerase to make the mRNA template of the protein. There is a physical relationship in a three-dimensional linear arrangement between the DNA of the operon, the normal closely applied histone molecules (generally less than 10,000 Daltons MW) about the DNA double helix, and the allogenetic repressor protein, typically 19,000 to 40,000 Daltons MW, sitting astride the DNA operator site, with its molecular structure displacing the histone from the area of the minor groove. From Rauka's model in 1966, and in agreement with Inoue and Ando's 1969 model of nucleoprotamine structure, the protamine, like histone, may occupy the minor groove of the DNA double helix, but also affect the binding sites of the major groove of Wilkin's 1956 model by charge cloud or physical interaction. Ando, T., Yamasaki, M., Suzuki, K., Protamines, Isolation, Characterization, Structure and Function. Molecular Biology, Biochemistry and Biophysics, V. 12, p. 81-84 (1973); and, Li, Hsueh Jei, Chromatin and Chromosome Subunits, pp. 159-161 (Academic Press, New York, 1977).
There is no excess histone in the free state in cells, due to highly toxic effects from the positive charge. Protamine, like histone, is a structural protein, but evolutionarily a protein of embryonic origin with nearly twice (K.sub.B =15.0M.sup.-1) the DNA binding coefficient of histone IV (K.sub.B =7.5M.sup.-1) at near physiological saline (0.154M aqueous NaCl). See, Table 1.
TABLE 1 ______________________________________ Binding of Basic Proteins to DNA.sup.1, Values of Binding Coefficient.sup.2, K.sub.B (M.sup.-1), in 0.1 M and 0.95 M NaCl Binding Coefficients (.times. 10.sup.2) In 0.1 M NaCl In 0.95 NaCl Maximum Binding Minimum Binding Native Denatured Native Denatured Protein DNA DNA DNA DNA ______________________________________ Protamine 15.0 5.9 1.2 0.6 Histone IV 7.5 5.1 1.8 1.3 Histone Ib 1.9 1.8 0.4 0.6 Poly-L-lysine 2.1 1.9 1.6 1.2 ______________________________________ .sup.1 Akinrimisi, E. D., J. Molecular Biology, "Binding of Basic Protein to DNA", 11, 128-136 (1965). ##STR1## - Protamine represents an early, evolutionary solution to the onslaught o allogenetic false inducers and false repressors.
After administered protamine-DNA complexes arrive in the repressed cell, there is a relative abundance of protamine, as compared with functional cellular DNA. Watters, C., Gullino, P., "Translocation of DNA from the Vascular into the Nuclear Compartment of Solid Mummary Tumors," Cancer Research 31, 1231-1243 (September 1971). The DNA in the adult cells is only protected by histones. The affinity of protamine for the DNA is, evolutionarily, greater than that of histone. The protamine dissociates from the allogenetic DNA and attaches to the open minor groove, due to its high binding affinity, replacing the lost histone and weakening the attachment of the allogenetic repressor. The allogenetic repressor is then displaced. DNAases in the cytoplasm attack the exposed allogenetic DNA, left instantaneously vacant by a protamine molecule in equilibrium, with the cell's DNA at the minor groove. The expelled allogenetic repressor is destroyed by circulating protease, or at least diffuses away from the major groove. Finally, the protamine is randomly, and slowly, replicated by histones. The cell's own repressors may now control the operator region, and transcription of DNA again. The cell returns to a normal genetic when a sufficient amount of allogenetic repressors are displaced to stop further uncontrolled transcription of unwanted proteins.
Displacement of allogenetic repressor by protamine interaction on the minor groove leads to normal translation of major groove base pairs.
The actual substitution of protamine follows a simple competitive inhibition model where the success of replacing the foreign repressor protein is directly proportional to a high protamine-DNA/foreign repressor ratio. The reaction is also influenced by the destruction of the allogenetic DNA of the original protamine-DNA complex, stopping the return of the protamine molecule to the allogenetic donor from the cell's heterogenetic DNA, thus, making the reaction irreversible.